Meningitis is either of bacterial or viral origin, the bacterial form being by far the most severe. The bacteria mainly responsible are Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae. Since the launch of a conjugate vaccine against H. influenzae type B (Hib), and its integration into routine infant vaccination, N. meningitidis is taking over as the leading cause of meningitis throughout the world, there being an estimated 2,600 cases per year in the USA alone.
The species N. meningitidis is subdivided into 13 serogroups according to the composition of the capsular polysaccharides. In addition, each serogroup is sub-classified into serotypes, subtypes, and immunotypes on the basis of other components of the bacteria. Three serogroups (A, B, and C) account for more than 90% of cases of meningitis, and in developed, industrial nations, serogroup B is responsible for 50 to 80% of cases.
Effective vaccines based on capsular polysaccharides exist to prevent meningitis caused by N. meningitidis serogroups A and C. The serogroup C polysaccharide vaccines do not produce a protective effect in children less than 2 years of age (the age range where there is the greatest risk of developing meningitis), however this drawback may be overcome by conjugating these polysaccharides to a carrier protein. Conjugation has the additional advantage of inducing an immunological memory against the antigen.
In contrast, the polysaccharide of N. meningitidis serogroup B displays little or no immunogenicity in man, irrespective of whether or not it is in a conjugated form. It would therefore be highly desirable to obtain a vaccine against neisserial disease induced by N. meningitidis (in particular of serogroup B) other than a polysaccharide-based vaccine.
A promising class of vaccine candidates are those using the outer membrane proteins (OMPs) of N. meningitidis, because they may provide antigens that are immunogenic and accessible to the human immune response. The OMPs responsible for the uptake of iron into the cell are particularly promising.
Iron is an essential nutrient for most bacteria. In the extracellular compartments of the human body iron is complexed mainly to transferrin in serum and to lactoferrin on mucosal surfaces (Finkelstein et al., 1983), with negligible amounts in the free form. Therefore, efficient iron acquisition is an important virulence factor for pathogenic bacteria. As regards N. meningitidis in particular (a strict pathogen of man), its iron requirements are met by using receptors for human iron-chelating proteins, such as transferrin or lactoferrin, which enable the cell to bind these proteins and thereafter to take up the iron needed for its growth. The synthesis of these receptor proteins is induced when the bacteria sense iron limitation.
The receptor proteins involved in the uptake of iron from transferrin, ThpA and TbpB (Cornelissen et al., 1992; Legrain et al., 1993; Anderson et al. 1994) and from lactoferrin binding protein A (LbpA) (Pettersson et al., 1993; 1994b; Biswas and Sparling, 1995) have been cloned and sequenced. The transferrin-binding receptor proteins form a complex in the outer membrane. In N. meningitidis, both ThpA and TbpB seem to be necessary for iron transportation (Irwin et al., 1993). ThpA is an integral membrane protein, whereas TbpB is a lipoprotein and is anchored to the membrane only with its lipid moiety. The current model for the mechanism of the receptor proposes that iron-loaded transferrin binds to the receptor complex. In this complex, the TbpB protein discriminates between ferrated- and apo-transferrin. Binding of transferrin results in the conformational change in the receptor, which releases iron from transferrin and opens a gated pore in ThpA, and iron can be transported accross the outer membrane (Cornelissen and Sparling, 1994; 1996).
The lactoferrin receptor is also thought to be an important virulence factor of N. meningitidis. The main site of entry into the human body is the nasopharynx, where lactoferrin is the main iron source. Furthermore, preliminary reports show that lactoferrin is able to cross the blood-brain barrier in acute inflammation (Gschwentner et al., 1997). It is possible that lactoferrin is also an important iron source for the meningococci at a later stage of infection, when the bacteria have reached the meninges. By using an affinity isolation procedure, a single lactoferrin-binding protein was originally identified (Schryvers and Morris, 1988). The structural gene for this receptor, designated LbpA, has been characterised (Pettersson et al., 1993; 1994b; Biswas and Sparling, 1995) and a topology model for the protein in the outer membrane has been proposed (Pettersson et al., 1994a). The protein shows homology to ThpA. In addition, part of a possible open reading frame was identified upstream of the lbpA gene, and the deduced amino acid sequence showed homology to TbpB (Pettersson et al., 1994a).
TbpB and other purified meningococcal OMPs have been the subject of previous patent applications with respect to their use as vaccines against N. meningitidis (e.g. TbpB, WO 9307172; 22 kDa surface protein, WO 9629412; haemoglobin receptor, WO 9612020; porin protein, WO 9503413; pilin proteins, WO 9408013; 64 kDa OMP, EP 474313-B I).
There is a need for identification and characterization of further members of the OMP family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, neisserial disease (for example meningitis).
Antibodies against LbpA do not seem to be bactericidal and it may therefore be of limited use as a vaccine candidate (Pettersson et al., 1993).
This invention identifies and characterises another lactoferrin-binding receptor protein, lactoferrin binding protein B (LbpB), its role in the utilisation of iron from lactoferrin, and its therapeutic uses.
There are several advantages LbpB has over the other OMP vaccine candidates. Firstly, in the blood-borne stage of meningococcal disease human lactoferrin is essential to the organism as it has a 300 fold greater affinity for binding iron than human transferrin, and hence the use of lactoferrin as an iron source is essential to the organism. Secondly, lactoferrin has a known antibacterial effect, and its concentration in the blood increases upon infection. It is also therefore important for the organism to bind human lactoferrin as a way of gaining some resistance to this effect. Lastly, human lactoferrin is the main source of iron to N. meningitidis at the place of entry of the bacteria to the human body (the nasopharynx).
The significance of these advantages is that LbpB antigens would be likely to be expressed at the cell surface in the vast majority of meningococci in the body, that the cell-surface domain of LbpB is likely to be very conserved because it must effectively bind lactoferrin, and that an immune response directed against the LbpB antigen may not only stop any infection of meningococcus in the blood, it may also stop even the carriage of the organism in the nasopharynx.